The Ashwell receptor is the major lectin of hepatocytes and rapidly clears from blood circulation glycoproteins bearing glycan ligands that include galactose and N-acetylgalactosamine. This asialoglycoprotein receptor activity remains a significant factor in the preparation and delivery of pharmaceuticals, yet a biological purpose of the Ashwell receptor has remained elusive. The ligands of the Ashwell receptor are endogenous glycoproteins and regulatory components in blood coagulation and thrombosis that include von Willebrand factor and platelets.
The liver controls the removal of exogenously administered glycoproteins from circulation as discovered by Ashwell and colleagues over 35 years ago (van den Hamer, C. J. A. et al., J. Biol. Chem., 245:4397-4402 (1970); Morell, A. G. et al., J. Biol. Chem., 246:1461-1467 (1971); Ashwell, G. and Morell, A., Adv. Enzymol. Rel. Areas Mol. Biol., 41:99-128 (1974); Hudgin, R. L. et al., J. Biol. Chem., 249:5536-5543 (1974)). These classical investigations identified the first vertebrate lectin as a hepatic receptor for glycoproteins bearing glycan chains that lack sialic acid, termed asialoglycoproteins (Ashwell, G. and Kawasaki, T., Methods Enzymol., 50:287-288 (1978); Ashwell, G. and Harford, J., Ann. Rev. Biochem., 51:531-534 (1982)).
The hepatic Ashwell receptor is one of multiple asialoglycoprotein receptors (ASGPRs) of the C-type lectin family and remains a fundamental consideration in design of clinical treatments to provide therapeutic levels of glycoproteins in circulation (Stockert, R. J., Physiol. Rev., 75:591-609 (1995); Weis, W. I. et al., Immunol. Rev., 163:19-34 (1998); Drickamer, K., Curr. Opin. Struct. Biol., 9:585-590 (1999)). ASGPRs of mammals mediate the capture and endocytosis of a wide range of exogenously administered glycoproteins with galactose (Gal) or N-acetylgalactosamine (GalNAc) residues at the termini of their glycan chains. More recent findings have indicated that some sialylated glycans are also ligands for the Ashwell receptor (Park, E. I. et al., Proc. Natl. Acad. Sci. USA, 102:17125-17129 (2005)). Localized to the vascular face of the hepatocyte cell surface, ASGPRs are positioned to remove and degrade potentially deleterious circulating glycoproteins (Ashwell, G. and Harford, J., Ann. Rev. Biochem., 51:531-534 (1982); Weigel, P. H., Bioessays, 16:519-524 (1994); Wahrenbrock, M. G. and Varki, A., Cancer Res., 66:2433-2441 (2006)). Nevertheless, the biological purpose of ASGPR activity has remained mysterious. Endogenous ligands have been difficult to identify and conservation of the Ashwell receptor throughout vertebrate evolution remains unexplained.
The Ashwell receptor is composed of type-2 transmembrane glycoproteins termed Asgr-1 and Asgr-2 that are encoded by distinct but closely linked genes, with variation in Asgr-2 structure due to RNA splicing (Drickamer, K. et al., J. Biol. Chem., 259:770-778 (1984); Halberg, D. F. et al., J. Biol. Chem., 262:9828-9838 (1987); Paietta, E. et al., J. Biol. Chem., 267:11078-11084 (1992)). Both Asgr-1 and Asgr-2 are highly conserved among mammalian species and may have originated from a single ancestral gene (Spiess, M. and Lodish, H. F., Proc. Natl. Acad. Sci. USA, 82:6465-6469 (1985); Hong, W. et al., Hepatology, 8:553-558 (1988); Takezawa, R. et al., Biochim. Biophys. Acta, 1171:220-222 (1993)). Although detectable in some extrahepatic tissues, the liver is the predominant site of their expression. Oligomerization of Asgr-1 and Asgr-2 has been observed in various cellular contexts with findings together supporting the possibility that Ashwell receptors may exist as Asgr-1/Asgr-2 hetero-oligomers, Asgr-1 trimers, and Asgr-2 dimers and tetramers, perhaps thereby altering substrate selectivity, binding affinities, and rates of endocytosis (Hardy, M. R. et al., Biochemistry, 24:22-28 (1985); Braiterman L. T. et al., J. Biol. Chem., 264:1682-1688 (1989); Henis, Y. I. et al., J. Cell Biol., 111:1409-1418 (1990); Bider, M. D. et al., J. Biol. Chem., 271:31996-32001 (1996); Ruiz, N. I. and Drickamer, K., Glycobiology, 6:551-559 (1996); Saxena, A. et al., J. Biol. Chem., 277:35297-35304 (2002); Weigel, P. H. et al., Biochim. Biophys. Acta., 1572:341-363 (2002); Yik, J. H. N. et al., J. Biol. Chem., 277:23076-23083 (2002)). Remarkably, while mice bearing reported null mutations in either Asgr-1 or Asgr-2 manifest decreased clearance of exogenous de-sialylated glycoproteins, they do not accumulate endogenous asialoglycoproteins in circulation, and lack significant intrinsic abnormalities (Ishibashi, S. et al., J. Biol. Chem., 269:27803-27806 (1994); Braun, J. R. et al., J. Biol. Chem., 271:21160 (1996); Tozawa R.-I. et al., J. Biol. Chem., 276:12624-12628 (2001)).
A genetic approach to disrupt the expression of sialyltransferases among intact animals has revealed endogenous glycoprotein ligands for one or more ASGPRs (Ellies L. G. et al., Proc. Natl. Acad. Sci. USA, 99:10042-10047 (2002)). Specifically, when ST3Gal-IV sialyltransferase activity is limiting or absent, ASGPR ligands are unmasked on a subset of regulatory and pro-thrombotic components of the mammalian blood coagulation system, including Von Willebrand Factor (VWF) and platelets. Mice lacking ST3Gal-IV show prolonged bleeding and coagulation times (Ellies L. G. et al., Proc. Natl. Acad. Sci. USA, 99:10042-10047 (2002)).
Disseminated intravascular coagulation (DIC) is a life-threatening coagulopathy involving the consumption of coagulation factors and platelets with the deposition of intravascular fibrin throughout the body resulting in multi-organ failure (World Health Organization, The Weekly Epidemiological Record, 14:110 (2003); Remick, D. G., Am. J. Pathol., 170:1435-1444 (2007)). As the small clots consume all the available coagulation factors and platelets, normal coagulation is disrupted and abnormal bleeding occurs. The small clots also disrupt normal blood flow to organs, which can result in organ failure and death.
Under normal hemostasis, the body maintains a balance between coagulation and fibrinolysis. The coagulation cascade yields thrombin that converts fibrinogen to fibrin, which forms a stable fibrin clot. The fibrinolytic system, which is activated in part by thrombin, then functions to break down the fibrin clot. Activation of the fibrinolytic system generates plasmin, which is responsible for the lysis of fibrin clots. In hemostasis, thrombin is the central proteolytic enzyme of coagulation and is also necessary for fibrinolysis.
In DIC, control of the processes of coagulation and fibrinolysis is lost, resulting in both widespread clotting and bleeding. DIC can be mediated by tissue factor (TF), which is released in response to exposure to cytokines and bacterial endotoxins. Upon activation, TF binds with coagulation factors that then trigger coagulation and an increase in circulating thrombin. At the same time, excess circulating thrombin leads to conversion of plasminogen to plasmin, resulting in fibrinolysis. The breakdown of clots results in excess amounts of fibrin degradation products (FDPs), which have powerful anticoagulant properties. Continued fibrin formation and fibrinolysis lead to hemorrhage from the consumption of coagulation factors and platelets and the anticoagulant effects of the FDPs.
DIC results from a number of clinical conditions, generally involving activation of systemic inflammation. Examples include, bacterial sepsis, viral infections, metastatic malignancies, and massive trauma.
The thrombohemorrhagic pathology associated with DIC continues to kill up to 50% of patients with severe septicemia in intensive care. In view of its life threatening nature, new methods for treating DIC are needed. This invention addresses these and other needs.